The rejection cascade in recipients sensitized to allo-Ag may trigger a form of an allograft loss that is mediated by memory T cells ("accelerated" rejection;AccR). To mimic the clinical scenario of sensitized patients, we have developed a murine cardiac AccR model in which B6 mice are challenged with B/c skin, and then rested for >40 days. When B/c hearts are transplanted into such sensitized mice, they are rejected in 4-5 days, with re-activated memory CD8 T cells playing the paramount role. The central tenet of this project is that graft TLR4-dependent pro-inflammatory innate response regulates the systemic CD8 T cell memory, and precludes successful organ engraftment in sensitized hosts. Aim 1: Determine the role of "positive" T cell costimulation in the development of alloreactive CD8 T cell memory, and its regulation by graft innate immune activation. Hypothesis: The activation of alloreactive memory CD8 T cells: 1/ proceeds via CD154- dependent direct and CD154-independent indirect mechanisms, and 2/ depends on TLR4-dependent inflammation at the graft site, which in turn critically affects the efficacy of T cell costimulation blockade. We will study costimulation requirements for memory CD8 re-activation, and dissect roles of ICOS, OX40, 4-1BB and CD27 signaling in alloreactive memory CD8 T cell generation. We will analyze the role of graft innate activation on alloreactive memory T cell activation. By utilizing mice with specific TLR4 mutations (MyD88/TRIF KO) as heart donors, we will determine: i/ how does local defective TLR signaling impact graft inflammation response; ii/ how does the absence of TLR4 affect systemic activation of alloreactive CD8+ T cells/graft infiltration by activated CD8 T cells;and iii/ the impact of deficient donor innate activation on host sensitization/efficacy of costimulation blockade. By using CD8+ TCR-tg (2C-tg) system, we will address the direct effects of T cell costimulation pathways on activation/differentiation of specific allo-CD8 T cells. Aim 2: Determine the role of "negative" PD-1 (CD279) - PD-L1/-L2 T cell costimulation in alloreactive CD8 T cell memory generation, and its regulation by graft innate activation. Hypothesis: The PD-1-PD-L pathways negatively regulate CD8 T cell memory activation by transmitting signals that limit their proliferation or trigger cell apoptosis. Harnessing physiological mechanisms of regulation by PD-1 on alloreactive T cells with PD-L on parenchyma cells depresses local CD8 memory T cells in a mechanism regulated by graft innate activation. First, we will evaluate how PD-1 pathway influences the primary CD8 T cell activation, and the memory repertoire. Second, we will differentiate the impact of negative signaling on CD4-dependent vs. -independent CD8 T cell memory recall. Third, we will define the role of PD-L on donor endothelial vs. BM derived cells (chimera). Fourth, we will explore mechanisms by which systemic vs. local PD-1-PD-L engagement (fusion protein/gene transfer) alters cardiac parenchyma inflammation/alloimmune responses. Fifth, we will study how intragraft innate immune activation regulates the PD-1-PD-L pathway in sensitized transplant recipients.